The productivity of a human adenovirus infection is dependent on the species of the cell which is infected. Cultured human cells support the full progression human adenovirus (Ad) infection, whereas rodent cells do not. For example, Ad12 DNA replication does not take place. The Ad12:hamster abortive system presents an opportunity to investigate how viral DNA replication is controlled during infection. Since some DNA replication-defective Ad mutants also exhibit altered transformation ability, the Ad12:hamster abortive system may, in the long run, provide insight into transformation events. Ad DNA replication may be divided into two processes--initiation and elongation-- each of which requires both viral and host proteins. One possible explanation for the block to Ad12 DNA replication in infected hamster cells is inactivity of hamster nuclear proteins in initiation and/or elongation. The first set of experiments in this proposal is designed to test whether hamster nuclear proteins are active for Ad12 DNA replication in vitro. The Ad12 replication will be optimized using the purified from infected human cells. The conditions for in vitro replication will be optimized using the purified viral proteins, exogenous viral DNA, and human nuclear extracts as the source of host proteins. The ability of hamster nuclear extracts to substitute for human extracts for both initiation and elongation will then be tested. Another possible explanation for blocked DNA replication is inactivity of one or more Ad12 replication proteins. The second set of experiments is designed to test whether (a) the low concentration, or (b) possible post-translational inactivation of the DNA-binding protein (DBP) could account for abortive infection. Purified DBP from infected human cells will be microinjected into infected hamster cells, and the cells analyzed by in situ hybridization to determine whether replication is triggered in vivo by the increased DBP concentration. In addition, DBP will be purified from infected hamster cells and tested for its activity for Ad12 DNA replication in vitro. The results obtained should add to our understanding of the influence of host species on the replication of viruses.